Recombinant coagulation Factor V/Va (FV/FVa)
Factor V/Va (FV/FVa) is a blood coagulation factor that enhances the efficacy of the serine protease Factor Xa by several orders of magnitude. It circulates the bloodstream in an inactive procofactor (FV), and upon cleavage, the procofactor transitions into an active cofactor, and assembles into the prothrombinase complex. This enzymatic complex converts the zymogen Prothrombin(II) into Thrombin(IIa) - the serine protease responsible for forming a fibrin plug at the site of a vascular injury. Previous studies have shown that a proteolytic cleavage of the procofactor activated the protein, but just the removal of a stretch (~50 aa) of basic residues was sufficient for the single chain factor V to be an active cofactor. Engineering a Factor V derivative that was single-chain and still had cofactor activity was crucial in establishing the idea that particular proteolysis was not necessary for factor V activation. In order to test the hypothesis that the basic region is crucial in keeping factor V as an inactive cofactor, the basic region was replaced with a non-homologous region of a related coagulation protein- Factor VIII. This new construct- despite it being a single chain with no proteolysis- this factor V construct was fully active as a cofactor in the prothrombinase complex. 1 Once the plasmid is established, site-directed mutagenesis can change all but one free cysteines for fluorescent labeling and systematic truncation of discrete regions can be manipulated by using primers to uncover the molecular mechanisms behind procofactor-cofactor transition. pED vectors In order to synthesized the recombinant factor V construct, a plasmid specific for expression in a mammalian system was used. pED vectors are useful because it allows for Unlike bacteria, mammalian mRNA is requires significant amount of processing. However, by placing the 5' untranslated regions of certain viruses (picovirus, encephalomyelocarditis virus) allow dicistronic mRNA, which with an internal open reading frame (ORF). This allows for the translations of both the protein of interest, and the ability to select the population of cells with a successful incorporation of the plasmid.2 Establishing a stable cell line Baby hamster kidney cells (BHK ) were co-transfected with a plasmid for G418 (a strong antibiotic) resistance, using a lipid-based transfection. Selection media with a given concentration of the antibiotic was used to select for cells that had both antibiotic resistance and factor V construct expression. Clones that expressed high amounts of factor V in the supernatant media (checked with a clotting assay for activity) were selected. The highest expressing clone is then expanded and seeded into triple flasks for purification. Purification Triple flasks (flasks with 3 layers) are inoculated with BHK cells that are stably expressing the factor V construct. Media collected was flowed onto a SP-sepharose (a negatively charged bead) by gravity flow, and eluted with high salt. Fractions were collected. Protein concentraion and activity of the fractions were determined by the UV spectrophotometer and clotting assay, respectively. The peak fractions are collected and flowed onto another SP sepharose column, using a FPLC. The protein was eluted with a calcium gradient, as factor V binds to the membrane using the Gla (gamma carboxy glutamate) domain, which coordinates calcium. As more calcium is added (and being coordinated by the Gla domain) only proteins that is binding to the column with a Gla domain will elute with increasing concentration of calcium. The fractions are checked for protein concentration and activity, and the peak fractions are pooled and concentrated, and frozen at -80 degrees. References Bunce MW, Bos MH, Krishnaswamy S, & Camire RM (2013) Restoring the procofactor state of factor Va-like variants by complementation with B-domain peptides. The Journal of biological chemistry 288(42):30151-30160.